Epigenetic abnormalities are very common in human cancers and play a critical role in tumorigenesis via dysregulation of gene expression and by affecting chromatin function. Inhibition of enzymes involved in epigenetic modifications has been recognized as a very attractive approach to develop novel drugs. Histone methyltransferases (HMTs) emerged as very promising class of anti-cancer drug targets and several recently developed inhibitors have been advanced to clinical trials, including inhibitors of DOT1L, EZH2 and PRMT5. These examples clearly emphasize significance of developing HMT inhibitors as novel anti-cancer drugs. In this project we propose to develop inhibitors of NSD1 histone methyltransferase. Translocation of NSD1 gene with Nup98 leading to Nup98-NSD1 fusion protein is found in pediatric acute myeloid leukemia patients with very poor prognosis. It has been validated that histone-methyltrasferase activity of NSD1 SET domain is required for oncogenic activity of Nup98-NSD1. To date, no NSD1 inhibitors have been described. We have identified small molecule compounds binding to NSD1 SET domain and employed extensive medicinal chemistry to significantly improve their activity. We developed potent cell-active NSD1 inhibitors blocking proliferation of Nup98-NSD1 cells. For our lead compound, we also demonstrated efficacy in animal model of Nup98-NSD1 leukemia. In this proposal, we will develop and optimize two classes of NSD1 inhibitors with two different mechanisms of action. We will optimize potency and drug-like properties of NSD1 inhibitors and perform extensive efficacy studies in animal models. In addition, our goal is to develop patient-derived xenograft (PDX) model of Nup98-NSD1 leukemia for efficacy studies. Our project will result in potent drug-like NSD1 inhibitors to be used as chemical probes and novel pharmacologic agents for acute leukemia.